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Renewable Energy 56 (2013) 85e89

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Renewable Energy

journal homepage: www.elsevier .com/locate/renene

Development of a membrane bioreactor for enzymatic hydrolysis of cellulose

Sulaiman Al-Zuhair*, Mohamed Al-Hosany, Yasser Zooba, Abdulla Al-Hammadi, Salem Al-KaabiChemical and Petroleum Engineering Department, UAE University, 17555 AlAin, United Arab Emirates

a r t i c l e i n f o

Article history:Received 24 May 2012Accepted 21 September 2012Available online 23 October 2012

Keywords:BioethanolCelluloseCellulasesMembrane bioreactor

* Corresponding author.E-mail address: s.alzuhair@uaeu.ac.ae (S. Al-Zuhai

0960-1481/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.renene.2012.09.044

a b s t r a c t

Cellulose hydrolysis is an important step in the production of bioethanol from lignocellulose. Usingenzymes, as a biocatalyst, is expected to have a lower utility cost compared to the conventional acidichydrolysis because it is carried out at milder conditions and does not require subsequent treatment step.The major obstacle to the practical realization of the potentials of enzymatic hydrolysis is the high cost ofthe enzymes and the slow reaction rate due to the inhibition of the enzyme by the products. In this work,a membrane bioreactor was simulated to tackle these two obstacles and enhance the reaction rate. It wasfound that for a 5000 kg h�1 lignocellulosic feed, to achieve 50% hydrolysis conversion, a 125 m3

membrane bioreactor containing 923 kg m�3 cellulase need to be used. The amount of the enzyme thatescapes from the system and needs replacement was estimated at 92 kg h�1. The membrane reactormodel was further tested using the competitive product inhibition model for the hydrolysis of totallyamorphous Carboxymethylcellulose (CMC). It was shown that the reactor volume required to achievea conversion of 50% was significantly less than that required for the lignocelluloses, even at a lowermembrane mass transfer coefficient.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Lignocellulosic biomass wastes have the capacity to producelarge quantities of liquid bioethanol, which can be used as a liquidbiofuel or as a source for hydrogen production. In order to breakdown the long chain cellulose into smaller fermentable simplesugars, a hydrolysis step is required. In hydrolysis the hydrogenbonds in the hemicellulose and cellulose are broken down toproduce pentoses and hexoses, respectively, which can both bethen fermented into bioethanol.

The most commonly applied methods of cellulose hydrolysis areclassified in two groups: chemical hydrolysis (dilute and concen-trated acid hydrolysis) and enzymatic hydrolysis [1]. Both enzy-matic and chemical hydrolysis require a pretreatment step toincrease the susceptibility of cellulosic materials to hydrolysis. Theutility cost of enzymatic hydrolysis is much lower compared to thealternative methods of acidic hydrolysis because it is carried out atmild conditions and does not require subsequent treatment step.Enzymes are also environment friendly, nontoxic and not corrosive[2,3]. The major obstacle to the practical realization of the poten-tials of enzymatic hydrolysis is the high cost of the enzymes and theslow reaction rate due to enzyme inhibition with the products [4].

r).

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The biocatalyst used for breaking down the cellulose is cellulase,which has three general forms working synergistically, namelyendoglucanase, exoglucanase and b-glucosidase [1]. Exoglucanaseattaches to one of the two ends of the cellulose molecule, andbreaks off one glucose unit at a time, and working toward thecenter. Endoglucanase attach anywhere along the polymer chainand breaks the chain into two separate chains. Both exoglucanaseand endoglucanase convert the cellulose to cellobiose, which isconverted to glucose by b-glucosidase [4]. It has been reported thatthe optimum temperature and pH for cellulose hydrolysis are40 �Ce50 �C and 4e5, respectively [5].

In order to tap into the great potential of cellulose derivedbiofuels, a reactor system that can handle the complex reaction andresult in an improvement in the rate and yield needs to be designedand optimized.

2. Membrane bioreactors

Using the enzyme in free form in a continuous process is usuallyunfeasible, which is mainly because the free enzyme leaves withthe effluent. A counter measure would be to recycle and reuse theenzyme. However, to achieve this, a protein separation unit isinevitably needed, which is an expensive process that renders theentire process unfeasible. On the other hand, by using the enzymein immobilized form, the enzyme is retained in the bioreactor andcan be reused, as long as the activity remains high. In addition, ithas also been reported that cellulase is susceptible to shear stress,

Steam Explosion

Settler1

1

23 3’

Settler2

4

5

6

7

8

9

10

11

CW

Fig. 1. Schematic diagram for the proposed MCSTR system.

S. Al-Zuhair et al. / Renewable Energy 56 (2013) 85e8986

which is a major concern as it leads to loss of enzyme activity [6].Immobilization of lipase provides a more rigid external backbonefor the enzyme molecule, allowing it to maintain its activity athigher stresses than if it is in free form.

However, using the enzyme in immobilized form restricts itsability to penetrate the solid substrate, and therefore the conver-sion is expected to be much less than in the case of using freeenzyme. To have a recyclable soluble enzyme without using theneed for expensive protein separation, membrane bioreactors canbe used to separate the glucose from the reaction medium throughthe membrane and then recycle the liquid outlet of the reactorwhich contains the enzyme using settler or centrifuge. Theunreacted cellulose and lignin in the reactor effluent stream can beseparated from the liquid phase containing the glucose ina conventional gravity settler. In order to recycle the cellulose,lignin should be separate from the solid mixture, using solventextraction. However, the use of organic solvent has many draw-backs, and since the raw material, waste biomass, is cheap it wouldbe cheaper if the unreacted cellulose and lignin are wasted.

As mentioned earlier, cellulases are inhibited by the products,glucose and cellobiose. Product inhibition of cellulases has longbeen known to significantly hinder the enzyme-catalyzed cellulosehydrolysis [2]. In general, for continuous biocatalytic reactions, CSTRis more suitable configuration for reactions subject to substrateinhibition, since the design allows minimization of the substrateconcentration. It was shown that by ignoring the inhibition, a CSTRwith a volume of only 31 m3 was needed to achieve 81% conversionfor flow rate of 1000 l h�1. However, when inhibition, which is themain drawback of cellulose hydrolysis, was taken into consideration,the required volume dramatically increased to 293 m3 [7].

In order to overcome the product inhibition, membrane biore-actor, where the reaction is coupled with continuous productsseparation, has been proposed. The molecular weight of glucose is180 g/mol, whereas that of most currently used fungal cellulasesranges from 35,000 to 65,000 g/mol [7]. Several studies usingvarious fungal cellulase systems and different cellulose substrateshave confirmed that it is possible, via membrane technology, toretain the enzymes present in the system while allowing thetransfer of low-molecular weight reaction products such as glucosethrough a membrane [7]. In addition to the positive effect ofseparating the product, the advantages of using the membranereactor include the ability of using the enzyme for a longer periodsof time and obtaining pure glucose in the permeate. There arehowever, some disadvantages of the system, which includepossible leaching of cellobiose in case of insufficient b-glucosidaserelative to cellulases in the enzyme mixture employed.

Gan et al. [8] proposed a dead end filtration cell, where a flatsheet Amicon polysulfone ultrafiltration membrane with a molec-ular weight cut-off (MWCO) value of 10,000 Da, was installed at thebase of the filtration cell. The system was operated in three strat-egies, namely batch system with continuous product separation,fed batch system with discontinuous product separation followedby the addition of substrate, and simultaneous reaction andproduct separation coupled with continuous feeding of substrate.The best production rate was achieved using the simultaneousreaction and product separation coupled with continuous feedingof fresh cellulose (MCSTR), which also provided constant concen-tration of sugars in the permeate.

2.1. Proposed membrane bioreactor system

The physicochemical structure and composition of lignocellu-loses hinder the hydrolysis of cellulose to sugars. Thereforea pretreatment step is essential to make the cellulose accessible tohydrolysis. In a successful pretreatment process lignin structure is

broken down and the crystalline structure of cellulose is disrupted,so that the enzymes can easily access and hydrolyze the cellulose.Steam Explosion is the most commonly applied pretreatmentprocess, owing to its low use of chemicals and limited energyconsumption [9]. In this method high pressure saturated water isinjected into a reactor charged with the biomass. During the steaminjection, the temperature rises to 160e260 �C. Subsequently,pressure is suddenly reduced and the biomass undergoes anexplosive decompression with hemicellulose degradation andlignin matrix disruption as a result. The process depends on resi-dence time, temperature, particle size and moisture content [10].

In the proposed cellulose hydrolysis system, the lignocellulosicraw materials are initially milled and then mixed with preheatedhigh pressure saturated water in the steam explosion reactor. Theeffluent of the steam explosion reactor is cooled down, usingcooling water, and then enters a settling tank to separate liquidmaterials, consisting of pentoses and water, from solid materials,consisting of the pretreated cellulose and lignin. The solid materialsthen enter the enzymatic hydrolysis membrane bioreactor, wherecellulose breaks down into glucose. Due to the advantages of theMCSTR, in this work similar bioreactor to that proposed by Ganet al. [8] has been used. However, instead of the dead end filtrationsystem, a jacketed cylindrical CSTR reactor with the vertical sidesmade of ultrafiltration membrane has been proposed. Theproduced glucose and water pass through the membrane to mixwith the aqueous stream from settler 1. The outlet of the reactor isintroduced to another settler to separate the supernatant, whichcontains the enzyme, from the solid consisting of unreactedcellulose and lignin. The supernatant stream is then recycled backto the reactor to allow the reuse the enzyme. The output of thereactor may then be fed into a fermentation unit to produce bio-ethanol. A schematic diagram of the system is illustrated in Fig. 1.

Before being fed to the steam explosion reactor, 5000 kg h�1

milled feed (Stream1), consisting of 58% cellulose, 22% hemi-celluloses and 20% lignin [11], is mixed with high pressure satu-rated water (Stream 2), with water: biomass ratio of 2:1 assuggested by Sengupta and Naskar [12]. In the steam explosionreaction, the lignocellulosic structure is broken and the hemi-celluloses are hydrolyzed to pentoses, presented as xylose. Thecomposition of hemicelluloses can be presented by (C5H10O5)n,where the n is in the range of 100e160. Taking 150 as a represen-tative value, the hydrolysis reaction of hemicellulose can then bedescribed by Eq. (1)

ðC5H8O4Þ150 þ 150H2O/150C5H10O5 (1)

S. Al-Zuhair et al. / Renewable Energy 56 (2013) 85e89 87

Assuming a first order dynamics and using the experimentaldata presented by Ballesteros et al. (2002) on herbaceous agricul-tural waste of 2e5 mm particle size at 210 �C and 4 bars, the rateconstant was found to be 0.274 h�1. Therefore, to achieve 85%conversion a residence time of 6.4 min per batch was found toneeded. The output stream from the steam explosion unit (Stream3), the composition of which is shown in Table 1, is cooled down to45 �C, which is the optimum enzymatic hydrolysis temperature,and then passed to Settler 1. It was assumed that 90% of the liquidcomponents in Stream 3 are separated in Stream 5, whereas 99% ofthe solid components are separated in Stream 4. The separatedliquid stream from Settler 2 (Stream 8), which contains majority ofthe enzyme, is combined with Stream 4 to form Stream 9 that is fedto the enzymatic hydrolysis bioreactor. It was assumed that 50%hydrolysis conversion is achieved, as suggested by Um [13]. It wasalso assumed that cellobiose did not accumulate inside the reac-tion, which can be achieved by using cellulose with high fraction ofb-glucosidase. The cellulose structure can be presented by(C6H12O6)n, where n ¼ 2500. The hydrolysis reaction of cellulosecan then be described by Eq. (2)

ðC6H10O5Þn þ nH2O/nC6H12O6 (2)

The glucose solution is then separated through membrane tocombine with Stream 5 to form Stream 10. It was assumed that 99%of the liquid components in Stream 7 are separated in Stream 8,whereas 99% of the solid components are separated in Stream 11.With the above assumptions, an Excel spreadsheet was constructedand iterative methodwas used to determine the compositions of allstreams. Table 1 shows the results when the separation of glucose iscomplete. The Excel spreadsheet could easily be used to find thecompositions for other glucose separations by simply changing thecell of the glucose in stream 10, all other compositions would thenautomatically be changed accordingly.

2.2. Membrane bioreactor design

The mathematic model proposed by Philippidis et al. [14] hasbeen adopted in this work. The model expressions given by Eqs. (3)and (4) were developed from mechanistic steps of celluloseconversion to cellobiose then to glucose, taking into considerationinhibition by both glucose and cellobiose.

r1 ¼ ky1 C

1þ BK1B

þ GK1G

(3)

r2 ¼ ky2 B

Km

�1þ G

K2G

�þ B

(4)

where, r1 and r2 are the rate of conversion of cellulose to cellobioseand cellobiose to glucose, respectively. C, B and G are the

Table 1Compositions of streams for complete separation of glucose.

Components Stream1 kg h�1

Stream2 kg h�1

Stream3 kg h�1

Stream4 kg h�1

Stream5 kg h�1

Cellulose 2900 0 2900 2871 29Lignin 1100 0 165 163 2Hemicellulose 1000 0 1000 990 10Water 0 10000 9873 987 8885Glucose 0 0 0 0 0Pentoses 0 0 1063 106 956Cellulase 0 0 0 0 0Total 5000 10000 15000 5118 9882

concentrations of cellulose, cellobiose and glucose, respectively. k1\

and k2\ are rate constants that depend on the enzyme concentration,

K1B and K1G are the inhibition constants of cellobiose and glucoseon reaction 1, respectively and K2G is the inhibition constant ofglucose on reaction 2. Km is Michaclis constant of b-glucosidase.The experimentally determined values of the parameters found inEqs. (3) and (4) are shown in Table 2. As shown, glucose inhibits thesecond reaction stronger because the inhibition constant K2G isvery small.

The concentration the cellulase components responsible forconverting cellulose to cellobiose, namely endo- and exoglucanase,was merged in the term k1

\ , whereas the concentration the cellulasecomponent responsible for converting cellobiose to gluclose,namely b-glucosidase was merged in the term k2

\ . The values of k1\

and k2\ , shown in Table 2, were found by Philippidis et al. [14] at

cellulose concentration of 25 IU g-cellulose�1. These values maychange depending on the activity of enzyme per gram of celluloseused in the reactor. The effect of b-glucosidase concentration on k2

\

was found to be linear, whereas the effect of cellulase concentrationon k1

\ and is not [14]. Nevertheless, for simplicity in this worka linear relationship has been assumed for both constants.

The system was assumed to be perfectly mixed, and thus, thematerial balance for each component at steady state can be pre-sented as shown in Eqs. (5)e(8),

FC ¼ FCi � r1V (5)

FB ¼ ðr1 � r2ÞV (6)

FG ¼ r2V � FPG (7)

FW ¼ FWi ��MWwater

MWGr1 �

MWwater

MWBðr1 � r2Þ

�V (8)

where, F represents the mass flow rate (kg h�1), which equals to theconcentration times volumetric flow rate, v (m3 h�1) and V are thevolume of the reactor (m3). The volumetric flow rate was taken asthe volumetric flow rate of water in the input stream to the reactor(Stream 9). FPG is the mass flow rates of glucose through themembrane, which can be described by Eq. (9)

FPG ¼ KmpðG� GPÞ (9)

where, Kmp is the mass transfer coefficient of the membrane(m3 h�1), G is the concentration of glucose in the reactor (kg m�3)and GP is the concentration of glucose in the permeate. Since theflow rate in the permeate was relatively high and had no glucose inits inlet, it was assumed that GP was very small compared to G.

The conversion, x, is defined as the amount of producedglucose over the total amount of inlet cellulose, which is describedby Eq. (10)

Stream6 kg h�1

Stream7 kg h�1

Stream8 kg h�1

Stream9 kg h�1

Stream10 kg h�1

Stream11 kg h�1

0 1443 14 2885 29 14280 165 2 165 2 1630 1000 10 1000 10 9900 97122 96151 97138 8885 9710 160 144 144 1443 160 0 0 106 106 0

92 9230 9138 9138 0 9292 109120 105459 110577 10475 3661

0

0.2

0.4

0.6

1500 750 100

Cel

lulo

se h

ydro

lysi

s co

nver

sion

, x

Membrane mass transfer coefficient, Kmp (m3/hr)

Fig. 2. Effect of the mass transfer coefficient of the membrane on the reactionconversion using a reactor volume of 125 m3.

Table 2Experimentally determined values of the rate parame-ters found in Eqs. (3) and (4).

Parameter Value

Philippidis et al. [14] modelk1\ (hr�1) 0.025

k2\ (kg m�3 h�1) 14.22

K1B (kg m�3) 5.85K1G (kg m�3) 53.16K2G (kg m�3) 0.62Km (kg m�3) 10.56Al-Zuhair et al [3] modelVmax (hr�1) 61.2Km (kg m�3) 0.03KiP (kg m�3) 169.0

S. Al-Zuhair et al. / Renewable Energy 56 (2013) 85e8988

x ¼ FPG þ FGFCi

(10)

Eqs. (5)e(10) were solved simultaneously using Excel spread-sheet to determine the volume required to achieve 50% conversionof cellulose. The nature of the reactions did not allow the iterativesolution of Excel to solve such them and the system did notconverged. For example, the first iteration at C ¼ 1000 kg m�3,G ¼ B ¼ 0, leads to very large r1 and r2 ¼ 0. The next iteration usingthe pervious value of r1 and r2 to find C, G and B, lead to small C (ornegative), a very large B and G¼ 0. Small Cmeans small r1 and largeB means large r2, this leads to very high value of G and very smallvalue of B in the next iteration and so on.

To overcome this problem, the initial guess should be the steadystate values. Least square method was used to minimize theobjective function, given by Eq. (11). The iteration was set tominimize the value of OF by changing the initial guesses usingsolver, with constrain that C, B and G should be positive numbers.

O:F: ¼ ðC1 � C2Þ2þðB1 � B2Þ2þðG1 � G2Þ2 (11)

where, the subscripts 1 and 2 represent trial and following trialresults, respectively. All the constants used were the same as thosedetermined by Philippidis et al. [14] and shown in Table 2, except k1\

and k2\ . Since larger amount of cellulose is used in this work,

compared to that used by Philippidis et al. [14], higher value of k1\

and k2\ were used. In the work of Philippidis et al. [14] k1

\ was0.025 h�1, which correspond to an activity of 25 IU g-cellulose�1. Inthis work, k1\ was taken as 0.95 h�1, which corresponds to cellulaseconcentration of 475 IU g-cellulose�1. Similar calculations weredone to k2

\ , taking its value to be 500 kg m�3 h�1 instead of14.22 kg m�3 h�1 and the activity of b-glucosidase was assumed tobe equal to the activity of cellulase.

The final results of this iterative technique using a reactorvolume of 125 m3 are shown in Table 3 and the effect of the masstransfer coefficient of the membrane on the reaction conversion isshown in Fig. 2. As expected, it was found that as the mass transfercoefficient increases, the conversion increases, and the product(glucose) amount in the permeate increases. The mass transfercoefficient has a strong effect on the permeate concentration, with

Table 3Effect of the mass transfer coefficient of the membrane on the flow rates in theoutput streams using a reactor volume of 125 m3.

Variable Kmp ¼ 1500 m3 h�1 Kmp ¼ 750 m3 h�1 Kmp ¼ 100 m3 h�1

FC (kg h�1) 1373 1408 1527FB (kg h�1) 59 84 170FG (kg h�1) 87 158 462Product (kg h�1) 1351 1220 712

more amount of glucose passing through as the mass transfercoefficient increases, leaving smaller amounts of glucose in thereaction media, which reduces the inhibition effect. This clearlyproves the viability of using the membrane reactor for theenhancement of cellulose hydrolysis. The mass transfer coefficientcan be controlled by choosing the type and porosity of themembrane.

The calculations were repeated using a mass transfer coefficientof the membrane of 1500 m3 h�1 for different values of reactorvolume and the results are shown in Table 4, and the effect ofreactor volume on the reaction conversion is shown in Fig. 3. Theresults show that the conversion increases as the reactor volumeincreases. It clearly seen that the reaction volume had a larger effecton the cellulose conversion, compared to the effect to the effect ofthe mass transfer coefficient shown in Fig. 2.

The activity of cellulase used was 710 IU g-cellulase�1 [15] fromwhich, the total weight of cellulase needed was determined to be27.7 tones, which correspond to a concentration of 923 kgm�3. Thisconcentration was then used in the material balance shown inTable 1 to determine the amount of make-up cellulase, which wasfound to be 92 kg h�1.

The membrane reactor model was further tested using thecompetitive product inhibition model developed by Al-Zuhair et al.[3], shown in Eq. (12),

d½P�dt

¼ Vmax½S�½S� þ Km

�1þ ½P�

KiP

� (12)

The values of the model parameters were determined for thehydrolysis of totally amorphous Carboxymethylcellulose (CMC) tobe as given in Table 2. Fig. 4 shows the effect of reactor volume onthe conversion of CMC hydrolysis using a mass transfer coefficientof the membrane of 100 m3 h�1. Since the hydrolysis of CMC ismuch simpler than lignocelluloses, the reactor volume required toachieve a conversion of 50%was found to be around 23m3, which is

Table 4Effect of reactor volume on the reaction conversion and flow rates in the outputstreams using a mass transfer coefficient of the membrane of 1500 m3 h�1.

Variable V ¼ 20 m3 V ¼ 60 m3 V ¼ 125 m3

FC (kg h�1) 2438 1885 1373FB (kg h�1) 55 63 59FG (kg h�1) 22.9 56 87Product (kg h�1) 354 867 1351

0

0.2

0.4

0.6

20 60 125

Cel

lulo

se h

ydro

lysi

s co

nver

sion

, x

Reactor volume, V (m3)

Fig. 3. Effect of reactor volume on the reaction conversion using a mass transfercoefficient of the membrane of 1500 m3 h�1.

0

0.2

0.4

0.6

5 10 15 20 25

CM

C h

ydro

lysi

s co

nver

sion

, x

Reactor volume, V (m3)

Fig. 4. Effect of reactor volume on the conversion of CMC hydrolysis using a masstransfer coefficient of the membrane of 100 m3 h�1.

S. Al-Zuhair et al. / Renewable Energy 56 (2013) 85e89 89

significantly less than that required for the lignocelluloses, even ata lower membrane mass transfer coefficient.

3. Conclusions

An enzymatic hydrolysis system has been proposed to simul-taneously solve the problems of reusing the enzyme, while keeping

it in soluble form, and removing the products which cause inhibi-tion. A CSTR bioreactor with membrane sides, where the productseparate through the walls, has been proposed. The effects of masstransfer coefficient through the membrane and reactor volumehave been assessed. It was found that for 5000 kg h�1 lignocellu-losic feed, to achieve 50% hydrolysis conversion, a 125 m3

membrane bioreactor containing 923 kg m�3 cellulase. Themembrane reactor model was further tested using the competitiveproduct inhibition model for the hydrolysis of totally amorphousCarboxymethylcellulose (CMC). It was shown that the reactorvolume required to achieve a conversion of 50% was significantlyless than that required for the lignocelluloses, even at a lowermembrane mass transfer coefficient.

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